1. Field of the Invention
The present invention relates to a purified and isolated merozoite protein derived by either conventional or recombinant means useful for the detection of Babesia equi in horses by means of a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA). The instant invention likewise relates to antibodies to the protein as well as cell lines which produce the antibodies.
2. Description of the Related Art
Equine babesiosis, caused by Babesia equi or Babesia caballi, is a tick-borne hemoprotozoan disease of horses (Schein, E., 1988. Equine Babesiosis, pp. 197-208. In M. Ristic (Ed.), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, Fla.). Clinical disease is characterized by fever, anemia, and icterus, most likely arising from hemolysis caused by merozoites, the intraerythrocytic stage of equine Babesia infection. Mortality rate is high during initial infection of horses introduced into enzootic regions, and horses which survive initial infection are protected from clinical disease upon subsequent challenge. It is hypothesized that this immunity acquired by horses in enzootic areas is the result of persistent infection.
The complement fixation test (CFT) is presently the official United States Department of Agriculture test for detecting antibody to B. equi and B. caballi. Horses with antibody to either parasite are restricted from importation into the United States. Three problems with the CFT are that (i) sera with anticomplement activity are not testable by the CFT; (it) sera which react with CFT control erythrocyte antigen cannot be evaluated by the GFT; and (iii) sera containing specific immunoglobulin G(T) [IgG(T)] antibody may yield false-negative results because IgG(T) does not fix complement by the classical pathway.
Merozoite surface proteins are known to be important in the pathogenesis of hemoprotozoan diseases because of their role in parasite recognition of, attachment to, and penetration of host erythrocytes. Antigens recognized by antibody from hosts demonstrating immunity to clinical disease during Plasmodium spp., B. rhodhaini, B. bovis, and B. bigemina infection include surface proteins of merozoites, the only blood stage of the parasite that is extracellular and directly accessible to serum antibody. It has previously been demonstrated that cattle immune to infection with B. bovis had high-titered antibody preferentially directed against four immunodominant merozoite surface proteins (Hines et al., Mol. Biochem. Parasitol. 37:1-9; 1989). Invasion of erythrocytes by merozoites of Plasmodium knowlesi was inhibited by immune sera, and inhibition of P. falciparum merozoite invasion of erythrocytes in vitro required high concentrations of specific antibodies. These observations suggest that antibody to merozoite surface proteins may block erythrocyte invasion in vivo and that these proteins should be tested as potential immunogens.
Detection of antibodies has been the method of choice for diagnosis of infection with equine Babesia spp.; however, the specificity or role of antibodies in the acquired protective immunity against clinical disease following equine Babesia infection has not thus far been determined.
Applicants have now developed a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) based on the use of a merozoite protein for detection of antibody to B. equi. The formatting of the CI ELISA overcomes the above three problems related to use of the CFT. Furthermore, a high concordance was found to exist between the CI ELISA and CFT in detecting antibody to B. equi.